The thyroid prohormone, thyroxine (T4), must be activated by 5'monodeiodination to 3, 5, 3'triiodothyronine (T3). This reaction is catalyzed by the Type 1 or 2 iodothyronine deiodinase (D1 or D2) and is the source of 80% of the T3 pool in humans. Both are down regulated during human illness causing the "low T3 syndrome". Type 3 deiodinase (D3) inactivates T4 and T3 by deiodination of the inner ring and may also increase in sick patients. Superficially, these enzymes are quite similar;all 3 are homodimers containing selenocysteine in a highly conserved active center and are integral membrane proteins. Nonetheless, each has unique catalytic mechanisms and regulatory processes which are essential to their different physiological roles. D1 in liver and kidney provides plasma T3 as well as inactivating T4 and T3 and their conjugates to preserve iodide. The D2 enzyme is highly efficient and expressed in human skeletal muscle, astrocytes, anterior pituitary, bone and brown adipose tissue. D2 provides substantial T3 to the plasma in humans and most of the T3 to the brain and stimulated brown adipose tissue. It is the D2 in the hypothalamic-pituitary axis that monitors circulating T4 and provides the key to T3 homeostasis during adaptation to iodine deficiency, an endemia affecting over 200 million of the world's population. Hypothalamic D2 is also increased in fasting and by endotoxins. In some populations, certain SNPs in the Dio2 gene have been associated with glucose intolerance, diabetes traits or hypertension. During development, D2 and D3 may change synchronously to modify the local tissue T3 concentration independent of circulating T3. Uncontrolled D3 over expression in large vascular tumors causes hypothyroidism in infants and adults. The first Aim of this proposal will evaluate the human deiodinases for those sequences required for homodimer formation using fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to understand the basis for their dimeric structure. The second Aim will address those factors which make D2 such an efficient deiodinase for T4 activation. We will identify the currently unknown putative thiol cofactor and show how it interacts with the active center using an in situ model system developed during the previous grant period. The third Aim will focus on the specific role of D2 in the hypothalamic-pituitary feedback axis as the sensor of plasma T4. Mice with pituitary-specific D2 inactivation will be created. Their TSH response to T4 during hypothyroidism or iodine deficiency will be compared with those of mice with D2 inactivated in both the pituitary and hypothalamus as well as with normal animals. This will define the role of pituitary vs hypothalamic D2 in the feedback regulation of thyroid function by T4. Our long term goal is to understand the regulation of peripheral thyroid hormone activation and inactivation enabling manipulation of these processes to increase T3 concentrations within specific tissues thereby enhancing basal energy utilization. Project Narrative: Thyroid hormone regulates energy expenditure in virtually every cell and is required for normal growth and cognitive development. By understanding the processes controlling its activation and inactivation in different tissues, we hope to exploit its potential for accelerating energy utilization to facilitate the maintenance of normal weight without producing adverse side effects.